In the prior art, there have been many attempts to improve producer strains and increase target metabolites using metabolic engineering approaches. However, the prior metabolic engineering methods for improving strains using molecular biological techniques required much higher cost and effort based on trial-and-error strategy. Recently, with the accumulation of genomic information and the development of various high-throughput screening techniques, methods capable of maximizing the production of useful substances in strains improved by metabolic engineering have been being developed. Particularly, as the entire genome sequences of various useful microorganisms have been identified, the construction of metabolic network models has become possible, thereby making substantial studies possible.
Thus, the use of the previously constructed metabolic network models made metabolic flux analysis possible under the assumption that “microorganisms undergo metabolic processes for the growth thereof (Varma et al., J. Theor. Biol., 165:503, 1993). As a result, the metabolic flux analysis can effectively provide the following, for example: (1) the identification of branch points in metabolic pathways; (2) the identification of substitute pathways; (3) the calculation of unmeasured external metabolites; and (4) the calculation of maximum theoretical yield (Stephanopoulos et al., Metabolic Engineering, Academic Press, NY, 309, 1998).
On the basis of the understanding of a huge amount of information provided recently, the development of new techniques for the development of effective producer strains is actively ongoing. By acquiring of the genome information of useful microorganisms by high-throughput screening techniques, metabolic network models were constructed (Edwards et al., Proc. Natl. Acad. Sci., 13:244, 2000; Foster et al., Genome Res., 13:244, 2003). Also, metabolic flux analysis methods for investigating integrated metabolic functions on the basis of metabolic network models were developed (Varma et al., J. Theor. Biol., 165:503, 2003; US 2002/0168654 A1). On the basis of these analysis methods, techniques capable of screening genes to be deleted based on metabolic network models so as to increase useful products of producer strains were developed. The Optknock method comprising screening genes to be deleted through the optimization of two axes consisting of flux for production and flux for growth and optimizing objective functions different from each other (Burgard et al., Biotechnol. Bioeng., 84:647, 2002, US 2004/0009466 A1), and the MOMA (minimization of metabolic adjustment) method capable of obtaining a partial optimal point by minimizing the migration of the optimum point caused by primary gene deletion of a strain having deletion of candidate gene from a wild-type strain was developed (Segre et al., Proc. Natl. Acad. Sci., 99:15112, 2002). On the basis of these methods, a gene screening method capable of screening genes to be sequentially deleted the first time, the second time and the third time was developed and actually applied for the production of lycopene (Alper et al., Metab. Eng., 7:155, 2004). In addition, a method for screening key metabolites increasing production yield of useful substances, comprising defining the metabolite utilization of a useful substance-producing organism as flux sum and perturbing the defined flux sum, and a method for improving a useful substance-producing organism by deleting and/or amplifying genes associated with said screened key metabolites were developed and applied for patent protection (Korean Patent Application No. 10-2005-62404).
In metabolic flux analysis, flux for production and flux for cell growth are generally in inverse proportion to each other (see FIG. 1). This indicates that, in the case of common microorganisms, the production of useful substances inhibits the growth of cells. For this reason, methods for increasing the production of useful substances through appropriate metabolic engineering techniques are required. Typical molecular biological approaches for achieving the metabolic engineering purpose include gene amplification techniques inducing the overexpression of target genes. Accordingly, for the improvement of organisms to improve the production of useful substances, the introduction of techniques for systematically amplifying genes and the development of techniques for applying the genes to organisms have recently been required in the field of art.